Hypoxia produces significant cellular perturbations in the pulmonary vasculature in lung diseases, such as the adult respiratory distress syndrome and chronic obstructive lung disease, where hypoxia is prevalent. These changes include a marked proliferation of smooth muscle cells (SMC) in the medial layer of the distal vessels and possible alterations in endothelial functions as suggested by increased platelet adhesion to the endothelium. Using cell culture systems, we have found that hypoxia produces a significant release from endothelial cell (EC) of hypoxanthine, a metabolite of ATP. This finding led us to investigate the effect of O2 tension on the enzyme xanthine oxidase/xanthine dehydrogenase (XO/XD) which catalyzes the conversion of hypoxanthine and xanthine to uric acid. Our preliminary results indicate that O2 tension might regulate the intracellular XO/XD activity of EC. Indeed, we found that intracellular XO/XD activity is absent when EC are exposed to hyperoxia and maximal (an approximate 300% increase compared to activity of EC exposed to normoxia) when EC are exposed to hypoxia or anoxia. The latter finding, combined with our previous report of an increased release of hypoxanthine from hypoxic EC, bears great significance in light of the recognized-potential for the hypoxanthine/xanthine oxidase system to produce reactive O2 species. Production of O2-based free radicals have, indeed, been shown to cause several deleterious effects on EC including cell injury and inactivation of the endothelial-derived-relaxing-factor (EDRF). We postulate that changes in the EC hypoxanthine/xanthine oxidase system in response to hypoxia may be a very early phenomenon that might be responsible for subsequent cellular changes in the pulmonary vasculature.
The Specific Aims of this project are to 1) further investigate changes in EC XO/XD in relation to O2 tension using a cDNA probe that we recently developed for the enzyme; 2) assess the potential for EC to produce reactive O2 species in response to changes in O2 tension; 3) examine possible cellular regulatory mechanisms of EC XO/XD in terms of transduction mechanisms, ion transport and second messengers; 4) examine the effects of selected cytokines and some known mediators of lung injury on XO/XD activity and mRNA expression; and 5) extend our cell culture work to in vivo studies by exposing rats to hypoxia and measuring XO/XD activity and mRNA expression of lung tissue.
